Substances such as insulin, chorionic gonadotropin, growth hormone, .alpha.-fetoprotein and immunoglobulin having antigenicity hereinafter referred to antigenic substances bind to the antibodies against these substances very specifically and very sensitively. Taking advantage of these features, numerous processes have been developed to measure the antigenic substances or their antibodies and many immunochemical measuring processes are already in practical use. To mention some examples, there are; immunodiffusion methods, in which the antigen and the antibody are caused to react with each other in agar gel; agglutination reaction and agglutination inhibition reaction methods, in which blood cells or fine particles like polystyrene latex are utilized as carrier of antigen or antibody; radioimmunoassay (RIA), in which radiosotopes are employed to label the antigen or antibody; enzyme immunoassay (EIA), in which enzymes are employed for the same purpose; and fluoroescence immunoassay, in which fluorescent materials are employed for the same purpose.
Meanwhile there is a competitive protein binding assay, in which a receptor protein or binding protein, i.e., a protein which specifically binds to the substance to be measured is utilized instead of the antibody in the immunochemical process.
These processes with respective characters are finding wide applications. Among others, RIA and EIA, which are far superior in the sensitivity of measurement and quantitative precision, are widely used, the substances measurable by them ranging from high molecular substances such as protein hormones, virus, immunoglobulins to low molecular ones such as peptides, steroids, synthetic medicines.
The principle, which is common to RIA and EIA, is characterized by two methods; competitive method and sandwich method.
The present specification concerns a case of the antigen as the substance to be measured and the antibody as the substance specifically bindable with said antigen. Not being confined to this case alone, however, the present invention is applicable equally to a case of the antibody as the substances to be measured and of the antigen as the bindable substance with said antibody; to an antigen-antibody system; and even to a physiologically active substance-receptor protein system.
Next, referring to a drawing, the competitive method and the sandwich method in RIA and in EIA are to be described.
As schematically illustrated in FIG. 1, the competitive method works such that an unknown amount of unlabeled antigen 1 and a given amount of labeled antigen 2 which is labeled by labeling agent are caused to react competitively with a given amount of the corresponding antibody 3. In this case, the unlabled antigen 1 and the labeled antigen 2 bind to the antibody 3 in proportion to their respective amounts and thereby the amount of the labeled antigen 2 to bind to the antibody 3 is reversely proportional to the amount of the unlabeled antigen 1. Next, by appropriate way the bound labeled antigen 2a and the free labeled antigen 2 are separated and the activity of the labeling agent in either of the two is measured. In the meantime a dilution series of reference substance, whose concentration is known, is prepared and in the same way described above the activity of labeling agent in each dilution is measured. A standard curve obtained by plotting the measured activities is utilized for determining the amount of the substance to be measured.
In the competitive method, the antigen is measured by causing the antigen to be measured and the labeled antigen to compete one with another for binding to the antibody. If the antibody used is one insolubilized, the antigen can be measured in the same way; moreover, the antibody too can be measured by causing the antibody to be measured and the insolubilized antibody to compete one with another for binding to the labeled antigen. U.S. Pat. Nos. 3,654,090 and 3,850,752 disclose the processes based on this principle. In these processes, the labeled substance directly binds to the insolubilized antibody, and this binding is competed by the binding of the substance to be measured to the insolubilized antibody and/or to labeled substance. Thus the amount of the labeled substance to bind to the insolubilized antibody is in reverse proportion to the amount of the substance to be measured.
Next the sandwich method works, as schematically illustrated in FIG. 2, such that an unknown amount of unlabeled antigen 1 to be measured is caused to react with the insolubilized antibody 4, i.e., an antibody to the antigen which has been insolubilized. Both come to bind to each other, yielding an antigen-body complex 5 (the first reaction). The complex 5 is separated from the reaction mixture and caused to react with a given amount of the labeled antibody 6 in which an antibody to antigen to be measured is bound to a labelling agent (the second reaction). The labeled antibody 6 binds to said complex 5, but a part of it which exceeds the bindability to the insoluble complex 5 remains in free state in the solution without binding to the insoluble complex 5. Next the bound labeled antibody 6a and the free labeled antibody 6 are separated and the activity of labeling agent in either of them is measured. In the meantime a dilution series of reference substance, whose concentration is known, is prepared and in the same way described above the activity of labeling agent in each dilution is measured. A standard curve obtained by plotting the measured activities is utilized for determining the amount of the substance to be measured.
In the sandwich method, since the labeled substance does not directly bind to the insolubilized antibody but does so through the substance to be measured, the amount of the labeled substance to bind to the insolubilized antibody will be proportional to the amount of the substance to be measured.
U.S. Pat. No. 3,791,932 discloses a process based on this principle.
Whereas in the competitive method a change in the activity of the labeling agent corresponding to increase or decrease of the amount of the antigen to be measured is little and accordingly with a mild slope of the standard curve the sensitivity of measurement is relatively low, in the sandwich method a change in the amount of the antigen to be measured directly corresponds to increase or decrease the activity of the labeling agent and accordingly with a steep slope of the standard curve the sensitivity of measurement is high. In the conventional sandwich method, however, in which after the first reaction between the antigen to be measured and the insolubilized antibody the antigen-antibody complex yielded has to be separated from the reaction mixture, and accordingly the upper limit of measurable amount of antigen is 10.sup.2 -10.sup.3 times the lower limit. Meanwhile, the upper limit of the amount of antigen to be measured may often amount to 10.sup.5 -10.sup.6 times the lower limit. For instance the blood level of .alpha.-fetoprotein is in normal humans less than 1.times.10.sup.1 ng/ml, but in a patient with primary hepatoma it may reach even 1.times.10.sup.6 ng/ml; in the latter case the specimens at several levels of dilution have to be prepared so that they can be made measurable by the process, whereby repeated dilution is likely to lower the precision of measurement.